Living systems comprise large polymeric structures made up of building blocks or components (monomers) which are themselves made up of elements. Examples of large polymeric structures found in living systems include proteins made up of amino acids, DNA and RNA made up of nucleic acids, complex carbohydrates made up of sugars, and lipids which can include fatty acids. The ability to measure the formation and degradation of polymers within cells or in cell-free systems is integral to understanding regulatory processes controlling cell proliferation and death, and the nature of chemical reactions. The biosynthesis and degradation of a polymer is particularly important to an understanding of various disease processes, development of an organism, cellular differentiation, tissue remodeling, and the like.
Existing methods used for determining polymer formation and degradation within cells or organisms are often referred to as “metabolic labeing” techniques. By far the most common current technique for the in vitro metabolic labeling of cells or tissues in culture uses 35S-methionine as a probe to measure the formation or degradation of cellular proteins. The technique of using radioactively-labeled amino acids as metabolic probes dates back to the late 1940's (Tarver, et al., J. Biol. Chem. 167:387-394 (1947)).
Although the technique is widely used for in vitro determinations of protein synthesis, there are major disadvantages to using radioactive amino acids or other exogenous (where the probe is not a usual constituent of the polymer being studied) radioactive probes in metabolic studies. These disadvantages include: 1) the danger to personnel using ionizing radiation, as well as the necessity to acquire permits for the purchase, storage, and disposal of radioactive materials; 2) the need to collect all of the protein synthesized to permit quantitative determinations since the method measures absolute levels of radioactive signal; 3) exogenous probe often alters the physical properties of the polymer being studied; 4) many elements exist for which there are no known radioactive species; and 5) use of radioactive probes is generally limited to in vitro systems or animal models since the concentration of probe needed for human studies usually exceeds legislated “safe-dose” standards.
These difficulties in the use of radioactive probes can be largely overcome as provided by the present invention by the use of a probe containing a stable isotope.
That most elements are mixtures of a number of stable isotopes has been known for over 60 years, and stable isotopes have been used as probes for metabolic studies within humans. The use of these probes was initially limited by their high cost and limited availability. Over the last few decades, many techniques for the production of stable isotope probes have been developed, and highly purified probes containing stable isotopes are commercially available. Further, instruments for the analysis of isotopic peaks (mass spectrometers) are now readily available to most workers in the field.
Use of a stable isotope for analyzing incorporation of a probe into biopolymers during in vivo metabolic studies has several problems. These problems include but are not limited to: 1) the need to breakdown the biopolymer of interest into smaller components (often amino acids); 2) the need to chemically derivatize the components followed by separating the components within their classes (often by gas chromatography); and 3) analyzing the mass of each component using a mass spectrometer (Halliday and Read, Proc. Nutr. Soc. 40:321-334 (1981)).
For each polymer of interest, the steps of separating the components within their classes and analyzing the mass of the components takes between 30 minutes to 1 hour. This greatly restricts the number of different polymers that can be analyzed on any one day. In addition, during the derivatization step, chemicals are added to the components, changing both the mass of the components and the isotope peak ratios in non-trivial ways (Lee et al., Biol. Mass Spectr. 20:451-458 (1991); Smith and Rennie, Biol. Clin. Endocrin. Metab. 10:469-495 (1996)). As stated by leaders in the field, “The technique is not without its disadvantages: the amino acid derivatization and quality control procedures are laborious and time-consuming; special instrumentation is required for precise GC-MS measurements; and a large number of time points are needed to accurately define the protein kinetics following a single-dose tracer administration.” Patterson. B. W., et al., J. Lipid Res. 32:1063-1072 (1991); see also, Goshe and Anderson, Anal. Biochem. 231:382-392 (1995)).
Despite the limitations inherent in the use and analysis of stable isotopes, widespread acceptance of gas chromatography and mass spectrometry have hindered the development of faster and simpler forms of analysis. As recently stated in a review of the field (Bier, Eur. J. Pediatr. 156(1):S2-S8 (1997)), “The worldwide acceptance and primacy of gas-chromatography-mass spectrometry (GCMS) as the preferred analytical tool for identification of abnormal metabolites . . . confirms the general nature of this method, supports the position that its limitations are relatively small in number, and attests to the nearly absolute specificity of metabolite identification using this method”.
The scientific and medical community's understanding of physiological processes is currently limited by the speed with which quantitative data can be collected, and the method by which the data is stored and processed in an easily retrievable fashion. Cobelli aptly summarizes this dilemma: “The principal difficulty attached to the mathematical analysis of physilogical and medical systems stems from the mismatch between the complexity of the processes in question and the limited data available from such systems.” Cobelli, et al., Am. J. Physiol. 246:R259-R266, 1984. Thus, there remains a need in the art to develop high-throughput techniques for measuring polymer synthesis in these complex systems. Quite surprisingly, the present invention fulfills this and other related needs.